Soybean is rich in protein and oil and serves as the most important legume crop globally. Soybean mosaic virus (SMV) is a severe threat to soybean production worldwide. MutMap, a gene-mapping technology based on map-based cloning and whole-genome resequencing, is utilized to clone key regulatory genes for agronomic traits in plants. However, no relevant studies have reported the cloning of genes resistant to SMV. We used an M3 mutant population derived from ethyl methanesulfonate mutagenesis of Williams 82, and conducted field inoculation experiments involving the SMV-SC3 strain. After field validation, two lines with high resistance to SMV were finally identified. Using MutMap, we initially screened candidate genes for SMV resistance and found that the G-to-A transitions of one candidate resistance gene, Glyma.13G194900, were at base positions 122 and 166. These transitions resulted in the substitution of glycine with glutamic acid (GGA→GAA) and valine with aspartic acid (GTT→GAT), respectively. Transgenic functional validation in soybean showed that the mutant allele of Glyma.13G194900 (designated Glyma.13G194900^M) substantially enhanced resistance to SMV-SC3, in contrast to the wild-type allele, which did not enhance resistance. Our results demonstrate that MutMap can rapidly identify SMV resistance-related genes to provide a genetic resource that accelerates the breeding of new SMV-resistant soybean.

Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold for their potential commercialization. Our previous study demonstrated that polypeptide fusion such as maize γ-zein or elastin-like polypeptide (ELP) could significantly increase the accumulation of foreign proteins. In the present study, a recombination strategy of polypeptide fusions (γ-zein or ELP) and suppression of intrinsic storage proteins (glycinin or conglycinin) via RNA interference was further exploited to improve the yield of the target protein in soybean seeds. Transgenic soybean plants harboring both polypeptide-fused green fluorescent protein (GFP) and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. The results showed that on both the glycinin and conglycinin suppression backgrounds, the average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of their counterparts without such suppressions in our previous study. Moreover, zein-GFP and GFP-ELP accumulation was also remarkably higher than unfused GFP on the glycinin suppression background. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase in the other. Additionally, the increases in the recombinant protein yield did not affect the total protein content and the protein/oil ratio in soybean seeds. Taken together, the results indicate that both the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins contributed to a higher accumulation of the recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds.

Background: Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold (1% of total soluble protein, TSP) for potential commercialization. Here, a recombination strategy of the polypeptide (maize γ-zein, or elastin-like polypeptide, ELP) fusions and suppression of endogenous storage proteins (glycinin or conglycinin) via RNA interference (RNAi) was exploited to improve yield of the target protein in soybean seeds. Results: Transgenic soybean plants harboring both the polypeptide-fused GFP and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. On the glycinin suppression background, average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of the unfused GFP, reaching to 2.69% and 2.30% TSP in the immature seeds, and 3.49% and 4.31 % TSP in the mature seeds, respectively. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase of the other. Moreover, the increases in the recombinant protein yield did not affect the total protein content or the protein/oil ratio in soybean seeds Conclusions: These results demonstrate that the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins can significantly increase the expression of recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds.